Heterokaryotic state of a point mutation (H249Y) in SDHB ...

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Title: Heterokaryotic state of a point mutation (H249Y) in SDHB protein drives

the evolution of thifluzamide resistance in Rhizoctonia solani

Running title: Evolution of thifluzamide resistance in R. solani

Author’s names:

Jianqiang Miaoa,b†, Wenjun Mub,c†, Yang Bib,d, Yanling Zhangc, Shaoliang Zhangb,

Jizhen Songc, Xili Liua,b*

aState Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant

Protection, Northwest A&F University, Yangling 712100, China

bDepartment of Plant Pathology, China Agricultural University, Beijing, China.

cKey Laboratory of Eco-environment and Leaf Tobacco Quality, Zhengzhou Tobacco

Research Institute of China National Tobacco Corporation, Zhengzhou, China.

dBeijing Key Laboratory of New Technology in Agricultural Application, Beijing

University of Agriculture, Beijing, China.

† These authors have contributed equally to this work.

*Correspondence to:

Xili Liu, State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant

This article is protected by copyright. All rights reserved.

http://dx.doi.org/10.1002/ps.6155




Protection, Northwest A&F University, Yangling 712100, China. E-mail:

[email protected]

Abstract

BACKGROUND: The sheath blight, caused by Rhizoctonia solani, can be

effectively controlled by the application of the succinate dehydrogenase inhibitor

(SDHI) thifluzamide. Although the resistant risk of thifluzamide in R. solani had been

reported, but the thifluzamide-resistance mechanism and the evolution of

thifluzamide-resistance in R. solani have not been investigated in detail.

RESULTS: No differences were found between the sequences of the SDHA, SDHC

and SDHD protein among the thifluzamide-sensitive isolates and the

thifluzamide-resistant mutants, but a single point mutation H249Y was found in

SDHB protein. Two different types of thifluzamide-resistant R. solani mutants were

characterized: homokaryotic type, carrying only the resistance allele, and

heterokaryotic type, retaining the wild-type allele in addition to the resistance allele.

The resistance level differed according to nuclear composition at position of codon

249 in sdhB gene. Molecular docking results suggested that the point mutation

(H249Y) might significantly altered the affinity of thifluzamide and SDHB protein.

Heterokaryotic mutants were able to evolve into a homokaryon when repeatedly


cultured on agar media or rice plants in the presence of thifluzamide, but thifluzamide

treatment had no effect on the genotypes of the homokaryotic mutants or the sensitive

isolates.

CONCLUSION: This study showed that H249Y in SDHB protein could cause

thifluzamide-resistance in R. solani. Fungicide application could promote

heterokaryotic mutants to evolve into a homokaryon.

1 INTRODUCTION

Rhizoctonia solani Kühn is a soil-borne pathogen that can cause economically

important diseases in a broad range of plants including vegetables, field crops, and

fruit and forest trees, as well as turf grasses and ornamental species grown throughout

the world.1-2 Disease symptoms can vary depending on the crop, from stalk rot in

cereals and corn, and damping-off in cotton and soybean, to black scurf in potatoes,

and root rot in sugar beet3 In rice, R. solani causes sheath blight and can infect all

stages of growth from seedling to heading, penetrating sheaths, leaves, and even

panicles and stems when the temperature and humidity are high enough. Diseased

plants are susceptible to lodging and produce reduced numbers of tillers and poorly

filled grains.4-5

R. solani can be categorized into 14 anastomosis groups (AGs) based on their pattern

of hyphal fusion (anastomosis), the dominant group globally being AG 1-IA.5-9


Although R. solani occasionally produces haploid basidiospores in the field, it

primarily exists as vegetative mycelia and asexual sclerotia.10 The hyphae of R. solani

lack septa and contain three or more nuclei per cell.6 Genetic exchange and the

formation of heterokaryons occur when the hyphae of two different isolates belonging

to the same AG.6,10,11 However, precise genetic studies regarding the reproductive

characteristics of R. solani are difficult to conduct, because the movement of nuclei

during anastomosis is difficult to control. Consequently, most investigations have

focused on haploid protoplasts rather than single basidiospores. Such studies have

confirmed that AG 1-IA can form both homokaryotic and heterokaryotic progeny.12 In

addition, changes to the protoplast-releasing procedure have been found to result in

protoplasts that contain different numbers of nuclei, making them genetically different

from their parental isolates.6

Although the antibiotic validamycin has been widely used to control soil-borne

pathogens such as R. solani, the control efficiency is somewhat reduced due to

resistance after years of use.13-15 Furthermore, the use of validamycin in Europe has

been prohibited in response to health and environmental concerns. Fortunately,

several other groups of fungicides can also provide effective control of the disease

caused by R. solani, including the benzimidazoles, triazoles, strobilurins, and

succinate dehydrogenase inhibitors (SDHIs).4, 16-18


SDHIs, which comprise 23 separate compounds, have been classified into eleven

groups based on their chemical structures (FRAC, https://www.frac.info/). Several of

the most recently discovered members having broad-spectrum activity against a wide

range of pathogens.19-22 The target site of SDHI fungicides is the succinate

dehydrogenase (SDH) complex, which plays an important role in the mitochondrial

tricarboxylic acid cycle, as well as being an important component of the respiratory

chain.23 The SDH complex is composed of four nuclear-encoded subunits, with two

subunits (flavoprotein subunit A, SDHA and iron-sulfur subunit, SDHB) forming the

membrane-peripheral domain, and two (integral membrane protein, SDHC and SDHD)

forming the membrane-anchor domain.24-25

Since SDHI fungicides have a single mode of action, they are vulnerable to the

development of resistance, and SDHI resistance has already been reported in several

basidiomycete and ascomycete fungi in both laboratory and field studies.21, 26-30

Several point mutations in the SDHB, SDHC, and SDHD subunits are now known to

cause SDHI resistance, the most common being associated with the conserved

histidine in the [3Fe-4S] center of SDHB (Table S1). However, reports had indicated

that an additional serine at position 83–84 of the SDHC subunit can also lead to

isopyrazam resistance in Fusarium graminearum,28 and a replacement of

phenylalanine by leucine at position 48 in the SDHC subunit was observed in


flutolanil-resistant R. solani isolates.31 Furthermore, non-target site SDHI resistance

has been confirmed in Zymoseptoria tritici, indicating that different mechanisms of

resistance associated with different SDHI fungicides can occur.32

In China, two SDHI fungicides, thifluzamide and flutolanil are firstly registered to

control rice sheath blight in 1999 and 1989 (www.chinapesticide.org.cn), respectively.

Thifluzamide, developed by Dow Agrosciences, belongs to the thiazole-carboxamide

group of SDHIs, and has excellent protective and curative activity against rice sheath

blight.5, 33, 34 Interestingly, although thifluzamide had been used for almost 21 years,

no field SDHIs-resistant R. solani isolates are detected in China according to some

recent studies.5, 35-38 According to our previous study, the risk of R. solani developing

resistance to thifluzamide is low to moderate,38 and a point mutation H249Y in SDHB

protein was found in thifluzamide-resistant R. solani isolates.39 Although R. solani

does not produce haploid asexual spores and sexual sporulation is very difficult to

induce, recombination through parasexuality or heterokaryon formation is possible.40

Under natural conditions, R. solani produces haploid sexual spores (basidiospores),

which can fuse to form heterokaryotic hyphae. The resulting multinucleate hyphae

can contain three or more nuclei per cell, which may have the potential for greater

genetic diversity and recombination than homokaryotic fungi. The resulting genetic

diversity could therefore facilitate the evolution of thifluzamide resistance in R. solani,


especially under strong selection pressures,41-42 although the evolution of fungicide

resistance in heterokaryotic fungi has not been studied in detail.

Based on our previous research, the aims of the current study were to (i) further

investigate and verify the molecular mechanism of thifluzamide resistance in R. solani

using molecular docking method, (ii) validate the probability of thifluzamide

resistance evolving from heterokaryotic isolates of R. solani in vitro and in planta.

2 MATERIALS AND METHODS

2.1 Isolates and culture conditions

Ten sensitive isolates collected from infected rice plants in the Jilin, Guangxi,

Guangdong, Fujian, and Shanghai provinces of China that had never been exposed to

thifluzamide and nine resistant mutants that were obtained in a previous study by

fungicide adaption or UV exposure were used.38 All the experimental isolates were

maintained by dark incubation on potato dextrose agar (PDA) at 25°C.

2.2 Fungicide sensitivity assays

The pure active ingredient thifluzamide (96%; Dow AgroSciences Company, China)

was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (1 × 105

μg/mL), which was stored at 4°C in the dark. Fungicide sensitivity assays were

conducted according to our previous study.38 Series of concentrations of thifluzamide

(0, 0.01, 0.025, 0.05, 01, and 0.5 μg/mL for sensitive isolates; 0, 0.2, 0.5, 1.5, 5, and


20 μg/mL for mutants with low to intermediate resistance; and 0, 0.5, 1.5, 5, 20, and

200 μg/mL for highly resistant mutants) were used.

2.3 Sequence and expression analysis of the sdh genes from R. solani

Genomic DNA was extracted using the FastDNA Plant Kit (Biomed Co. Ltd, Beijing,

China), and total RNA extracted using the SV Total RNA Isolation System (Promega

Corp., Beijing, China) according to the protocol of the manufacturers. The resulting

RNA was then reverse transcribed to cDNA using the EasyScript Reverse

Transcriptase Kit (TransGen Biotech, Beijing, China).

Primer sets (Table S2) were designed to amplify full-length sequences of the sdh

genes using BioEdit v7.0.9 (Ibis Biosciences, USA) and DNAMAN software 6.0

(Lynnon BioSoft, Quebec, Canada) in conjunction with data from the draft genome of

R. solani AG-3 (http://www.rsolani.org) and the sdh sequences from other species

including Coprinus cinereus, Ustilago maydis, Magnaporthe grisea, Botrytis cinerea,

Escherichia coli, Homo sapiens, and Gallus gallus (Table S3). The PCR was

performed using Taq PCR MasterMix (TransGen Biotech, Beijing, China), and

processed in a MyCyclerTM thermocycler (Bio-Rad, California, USA). All the PCR

products generated in the study were sequenced by Invitrogen Life Technologies

(Beijing, China) and amino acid sequences deduced from the cDNA sequences using

the DNAMAN software package. Introns were then removed from the R. solani


sequences using data from the C. cinereus sdh genes and the software hosted on the

Softberry website (http://linux1.softberry.com/berry.phtml).

The real-time qPCR was performed using the ABI7500 sequence detection system

(Applied Biosystems, Warrington, UK) and the SYBR Premix Dimer Eraser kit

(Takara Biotechnology Co.,Ltd., Dalian, China) with the primers listed in TableS3 and

the protocol of the manufacturer. The relative quantities of the PCR products were

calculated using the 2-ΔΔCt method and the actin gene as a reference to normalize the

quantification of sdhB expression. The entire experiment was conducted three times.

2.4 Molecular docking of thifluzamide in the SDH complex of R. solani

For docking studies of thifluzamide in the SDH complex, the crystal structure of SDH

complex II from Escherichia coli (PDB code 2WDQ), which exhibits 45% sequence

identity with the sequence from R. solani with all of the key amino acids being highly

conserved, was used as the template.43 The binding conformation of carboxin in the E.

coli complex II produced docking results with a good overlap to that of the crystal

structure of 2WDQ, and a root mean square deviation of 0.51, which indicated that it

was a suitable model for the current study. The molecular docking of thifluzamide in

the quinone-binding pocket of the R. solani SDH complex was investigated using

Sybyl 7.3 software. To prepare the protein structure for docking, the crystal of SDH

complex II from E. coli (PDB code 2WDQ) was firstly downloaded from PDB


database (http://www.rcsb.org/). Then, addition of hydrogen atoms, side-chain

protonation states, and adjustment of the rotational states of histidine, glutamine,

asparagine residues were conducted to produce the docking model. In addition, the

water near the binding pocket (between SDHB_S161 and SDHB_H207) was kept in

the model as several hydrogen bonds were mediated by this water molecule. The

binding-pocket receptor and thifluzamide were energy minimized using the MMFF94

electric charge and the Tripos force field; the energy value was less than 0.005

kcal/mol/Å, and the cycle index 1000. Having established that these parameters were

suitable, both thifluzamide and carboxin were docked into the binding-pocket using

the Surflex-dock module of the Sybyl 7.3 software. The histidine at position 207 of

the sdhB gene from E. coli (the analogous position 249 in sdhB gene from R. solani)

was then site-directed mutated to tyrosine with the Biopolymer-Replace Sequence

module to investigate how the corresponding point mutation of the histidine residue at

position 249 in the SDH of R. solani affects thifluzamide binding. The amino acids

within approximately 10 Å of the point mutation were optimized using the MMFF94

electric charge and Tripos force field with the Dynamics module of the Sybyl 7.3

software package.

2.5 Production and regeneration of protoplasts

The thifluzamide-sensitive isolate JHT158-3 (homokaryotic for C at 975 of the sdhB


gene) and the highly resistant mutant T2 (homokaryotic for T at 975 of the sdhB gene),

were paired on PDA plates and dark-incubated at 25°C for two days. Ten mycelial

plugs (5 mm in diameter) were then cut from the intersection of the two colonies and

used to inoculate potato dextrose broth (PDB). After 24 hours of dark incubation at

25°C with shaking (120 rpm), the mycelium was removed with forceps, washed in an

osmotic medium (0.98 M MgSO4, 8.4 mM Na2HPO4, 1.6 mM NaH2PO4),44 and

ground with a sterile pestle and mortar before being transferred to a 250-mL flask

containing 100 mL CM medium (5 g/L glucose, 5 g/L malt extract, 5 g/L yeast

extract),12 and dark-incubated at 25°C with shaking (120 rpm) for 24 hours. The

mycelium was then collected by centrifugation at 1500 rpm for 10 minutes. After the

supernatant had been removed, the mycelium was washed in 10 mL osmotic medium

and centrifuged as before. The supernatant was again removed and the fungal cell

wall digested using 15 mL of an enzyme mixture (20 mg/mL snailase, Biodee, China;

20 mg/mL cellulase and 20 mg/mL lysing enzymes, Sigma Chemical, China), which

had been dissolved in STC (1.0 M sorbitol, 10 mM Tris-HCl, and 50 mM CaCl2)44

using a magnetic stirrer, centrifuged at 8,000 rpm and passed through a 0.22-μm filter.

After four hours of incubation at 28°C with shaking (60 rpm), the undigested

mycelium was removed by passing the preparation through two layers of Miracloth

and washing with STC. The lysates containing the protoplasts were then centrifuged


at 4,000 rpm for 10 minutes and resuspended in STC to a final concentration of 104

protoplasts/mL. The resulting suspension was used to inoculate a solid RM medium

(200 g/L potato, 18 g/L dextrose, 182.17 g/L mannitol, 14 g/L agar) in 200-μL

aliquots and dark incubated at 25°C for two to three days, before the hyphal tips

derived from individual protoplasts were subcultured onto fresh PDA plates using a

sterile knife.

2.6 Pyrosequencing assays

The pyrosequencing assays in the current study were performed using the PSQ 96MA

system (Gene Limited Company, Shanghai, China). Biotin-labeled samples were

prepared by PCR using 40-μL reaction mixtures containing 2 μL of template DNA or

cDNA, 4 μL 10×PCR buffer, 0.8 μL dNTP (10 mM), 0.3 μL of each primer (10 μM),

and 0.4 μL Takara Hot Start Taq. The PB-F1/PB-R1-Bio primer set was used for the

amplification of genomic DNA (Table S2). The PCR was processed in a MyCyclerTM

thermocycler (Bio-Rad, California, USA). The biotinylated PCR products were then

immobilized on streptavidin-coated Sepharose beads and washed with 70% ethanol

before being sequenced using a PyroMark ID pyrosequencer with PSQ 96MA

software and 0.5 μM sequencing primer (Table S2).

2.7 The potential for thifluzamide resistance to develop in R. solani: in vitro and

in planta experiments


The in vitro tests were conducted by repeated subculture (20 in total) of the parental

isolates JHT158-3 and T2, and the nine protoplast-regenerated isolates on PDA

containing either 0 or 0.05 μg/mL thifluzamide (the approximate EC50 value for

thifluzamide in field isolates of R. solani). Each subculture was made after three days

of dark incubation at 25°C by scraping the mycelia from the original plates onto fresh

media to produce the next generation. The DNA from the first, fifth, 10th, 15th, and

20th generation were extracted for the pyrosequencing analysis described above. The

experiment was performed twice.

A similar experiment was conducted by subculturing the same isolates on rice plants

under greenhouse conditions. The initial inoculations were made using mycelial plugs

(7 mm), which were cut from four-day PDA cultures and placed beneath the leaf

sheath of rice plants (cultivar Jasmine 85) at the six to seven leaf stage. Thirty rice

plants were inoculated with each isolate. The inoculated leaf sheaths were then

wrapped in aluminum foil. After seven days under greenhouse conditions (28°C

maintained at >80% humidity), when typical lesions had formed on the inoculated

plants, the aluminum foil was removed and the plants were sprayed with the

commercial thifluzamide formulation Pulsor (24% thifluzamide suspension

concentrate) at a rate of 100.67 µg/mL (the minimum recommended field dose), using

a larynx sprayer to ensure that the fungicide was dispersed uniformly on each plant.


Negative control treatments were prepared by spraying inoculated plants with water.

After two days, one small lesion was cut randomly from the plants in each treatment

and surface sterilized in NaClO for fine minutes before being placed on PDA

containing 50 μg/mL of both penicillin and streptomycin. Each sample was then

dark-incubated at 25°C for three days to produce the inoculum for the successive

subculture and the DNA samples for the pyrosequencing analysis described above.

The experiment was performed twice.

2.8 Fitness of heterokaryotic thifluzamide-resistant isolates of R. solani

The fitness of 14 R. solani was assessed using several criteria including in vitro

mycelial growth and biomass, in vitro sclerotia production and germination, and in

vivo virulence according to our previous study.38

2.9 Statistical analysis

The data collected in the study were analyzed using Statistical Analysis System

software (version 9; SAS Inc., Cary, NC). The EC50 values for each isolate were

calculated by linear regression using PROC REG, and Fischer’s least significant

difference test to assess significant among the different treatments of the fitness tests.

3 RESULTS

3.1 Mutations in SDH proteins associated with thifluzamide resistance

The full-length and complete coding sequence of sdhA, sdhB, sdhC, sdhD were


app:ds:streptomycin

identified and analyzed (Fig. S1) in ten thifluzamide-sensitive wild-type isolates, and

nine thifluzamide-resistant mutants obtained in our previous study.38 No differences

were found between the sequences of the SDHA, SDHC and SDHD protein among

the thifluzamide-sensitive isolates and the thifluzamide-resistant mutants, but a single

point mutation was found in the sdhB gene of all the resistant mutants, which resulted

in the histidine residue (CAT) at codon 249 being replaced by a tyrosine residue (TAT)

in SDHB protein. Furthermore, the results of overlapping peaks produced during the

Sanger sequencing indicated that the mutants with low to intermediate resistance were

heterokaryotic (containing both CAT and TAT codons dislike just the CAT codon in

the sensitive isolates), while the highly resistant mutants were homokaryotic,

containing only the TAT codon (Table 1, Fig. S2). Real-time qPCR results indicated

no overexpression of the sdhB gene when mutants were treated with thifluzamide at

concentrations corresponding to the EC50 and EC90 of thifluzamide (Fig. 1).

3.2 Thifluzamide docking in the SDH complex of R. solani

The docking score of 6.03 indicated that the binding energy of carboxin was similar to

that of thifluzamide. The key interactions suggested that four direct hydrogen bonds

were formed between thifluzamide and residues SDHB_S161, SDHB_W164,

SDHC_R31, and SDHD_Y83 of E. coli, while two hydrogen bonds (less than

classical hydrogen bond distance) mediated by water molecules were formed,


including one between hydrogen (No. 1) and one of thifluzamide’s fluorines, and

another one between hydrogen (No. 2) and the oxygen of SDHB_S161 (Fig. 2A). The

results also suggested that a salt bridge was formed between SDHB_H207 and the

heme prosthetic group (Fig. 2A).

The effect of changes to SDHB_H207 (the analogous SDHB_H249 in R. solani) on

the affinity of the SDH binding pocket for thifluzamide was investigated by the

introduction of Y207 instead of H207 in the E. coli SDH complex II. The resulting

docking score of 3.83 was almost two orders of magnitude lower than that of the

wild-type protein, suggesting that reduced affinity was therefore responsible for the

reduced thifluzamide sensitivity of the mutants. The corresponding docking image

indicated that the mutation SDHB_H207Y (the analogous SDHB_H249Y in R. solani)

reduced the number of hydrogen bonds between H2O and thifluzamide (Fig. 2B). This

point mutation (SDHB_H207Y) also changed the position and orientation of the water,

and caused the ligand’s center-of-mass moved and the distance is greater than the

classical hydrogen bond distance. This change caused the loss of the hydrogen bond

between thifluzamide and SDHD_Y83 (Fig. 2B). The mutation also broke the salt

bridge between the heme prosthetic group and thifluzamide. These changes to the key

interactions of SDHB and thifluzamide indicated that the mutation significantly

altered the docking of thifluzamide (Fig. 2B).


3.3 Pyrosequencing for the rapid detection of thifluzamide-resistant R. solani

isolates with a point mutation H249Y in the sdhB gene

The accuracy of the Pyrosequencing used in current study was confirmed using

regression analysis of standardized DNA mixtures (pEASY®-T1 cloning vectors

containing the full-length of sdhB gene from thifluzamide-sensitive isolate JHT158-3

or thifluzamide-resistant isolate T2). The measured mutation allele frequency (T in

975 of sdhB) using Pyrosequencing and the expected frequency showed good linear

relationship (Fig. 3). Then, a total 207 R. solani isolates with different thifluzamide

sensitivity were determined using Pyrosequencing, and the frequency of T in 975 of

sdhB gene showed a positive correlation with the thifluzamide sensitivity (Fig. S3).

3.4 Evolution of thifluzamide resistance in heterokaryotic mutants of R. solani in

vitro and in vivo

The genetic background of each isolate was investigated using protoplasts derived

from hyphal fusions between the sensitive isolate JHT158-3 and the resistant isolate

T2. Eight single protoplast isolates were selected for evolution experiment (Table 1).

Sanger-sequencing and Pyrosequencing analysis results showed that P97 had only

C975 which was similar to JHT158-3, while P48 contained only T975. The other six

mutants (P37, P45, P196, P216, P229, P232), which showed moderate resistance and

contained two alleles that encoded either histidine or tyrosine at position 249 (Table


1).

Then, the eight mutants were subjected to repeated subculture in either the absence or

presence of thifluzamide in vitro, and the T/C ratio of the isolates were measured after

the first, fifth, 10th, 15th, and 20th subcultures. All the highly resistant and sensitive

isolates remained homokaryotic. However, the heterokaryotic mutants P229 changed

to be homokaryotic for the mutated allele after just five subcultures on PDA

containing 0.05 μg/mL thifluzamide (Fig. 4A, 4B).

Seven resistant mutants (P48, P37, P45, P196, P216, P229, P232) were also subjected

to repeated subculture in either the absence or presence of thifluzamide in vivo, and

the T/C ratio of the isolates were measured after the first, third, fifth, seventh, and 10th

subcultures. P216 becoming homokaryotic after the fifth generation when sprayed

with the recommended dose (100.67 µg/mL) of the commercial thifluzamide

formulation Pulsor (Fig. 4C, D).

3.5 Fitness of the parasexual progeny of thifluzamide-resistant mutants

In general, the mycelial growth, mycelial biomass, sclerotium weight and sclerotium

germination of the isolates containing the thifluzamide-resistance allele were similar

to or lower than that of the sensitive isolates (Table 2). Consequently, the compound

fitness index (CFI), which collated all the fitness parameters into a single value, was

lower for most of the isolates bearing the thifluzamide-resistance allele than that of


the sensitive isolates, except for P229 and P196, the CFI value of which was

significantly higher and comparable to that of parental isolate JHT158-3 (Table 2).

4 DISCUSSION

Many studies had shown that point mutations in the SDHB, SDHC, and SDHD

subunits of various fungi can lead to resistance to SDHI fungicides (Table S1). The

current study was initiated to discover whether the thifluzamide-resistant R. solani

mutants identified in our preliminary study34 utilized a similar mechanism of

resistance. The deduced amino acid sequences of the cloned sdhA, sdhB, sdhC, and

sdhD genes, which encode the flavoprotein (Fp), the iron-sulfur protein (Ip), and the

large and small membrane-anchor proteins SDHC and SDHD, respectively, were

found to have a high degree of similarity—97%, 90%, 96%, and 94%—with the

SDHA, SDHB, SDHC, and SDHD sequences of R. solani AGI-IB listed on the NCBI

website. Comparison of the novel R. solani sequences with those of other

basidiomycetes, ascomycetes, bacteria, and animals revealed that the SDHA subunit

exhibited the highest degree of conservation (74%), followed by SDHB (64%), SDHC

(43%), and SDHD (43%). These results were consistent with other studies that have

found the Fp and Ip to be more highly conserved than the two membrane-anchor

subunits.45-46

No nonsynonymous point mutations were found in SDHA, SDHC, and SDHD


subunits from the resistant mutants, and only H249Y was identified in their SDHB

subunit. Considering that real-time PCR analysis displayed no overexpression of the

sdhB gene in any kinds of the mutants, and H249Y in SDHB protein has also been

reported in other fungi with SDHI fungicdes resistance (Table S1), it is highly likely

that this point mutation is responsible for the thifluzamide resistance observed in the

R. solani mutants.

The results of the docking analysis for thifluzamide in the quinone-binding pocket of

the SDH complex supported the resistance mechanism. Similar to the docking of

carboxin in the crystal of the E. coli SDH complex,43 several key residues from the

SDHB, SDHC, and SDHD subunits, including SDHB_H207Y (the corresponding

SDHB_H249Y in R. solani), were directly involved in thifluzamide binding.

Furthermore, the results showed that the SDHB_H207Y mutation weakened the

interaction with thifluzamide due to conformational changes associated with the loss

of the water-mediated hydrogen bond between the now-absent histidine residue and

S161, and the loss of the salt bridge with the heme prosthetic group. These results

were consistent with other studies that have also found similar mutations can change

the docking of other SDHI fungicides to the quinone-binding pocket of the SDH

complex.21, 27, 47 The negative impact of the SDHB_H249Y point mutation on the

interaction between thifluzamide and the binding pocket of the SDH complex in R.


solani support that this mutation is responsible for the observed thifluzamide

resistance of the mutant isolates. A mutation of SDHB_H267Y (the corresponding

SDHB_H249Y in R. solani) could lead to high in vitro resistance factors for boscalid

(RF= 473.8), isopyrazam (RF =142.6), and carboxin (RF = 65.9) in Mycosphaerella

graminicola, which was also verified by molecular docking.27 SDHB_H267Y lead to

aromatic sidechain makes edge to face stacking to the SDHIs and the tyrosine

hydroxyl group a hydrogen bond to SDHD_D129. A direct hydrogen bond of the

tyrosine to the hydrogen bond accepting groups of boscalid, isopyrazam, and carboxin

is unlikely potentially impairing the binding of these molecules and thereby

explaining the higher resistance factors observed for these molecules in M.

graminicola.27

Further examination revealed that the degree of thifluzamide resistance was related to

the nuclear composition of the mutants, with homokaryotic mutants exhibiting much

higher resistance than their heterokaryotic counterparts, which still possessed

wild-type versions of the sdhB allele. The relationship between nuclear composition

and fungicide sensitivity is important because R. solani readily undergoes

anastomosis, and therefore nuclei bearing resistance mutations can readily be

transmitted to sensitive strains. These characteristics result in a high potential for the

recombination and selection of different alleles, including those associated with


fungicide resistance. In the context of the current study, it seems likely that the low to

intermediate level of thifluzamide resistance observed in the heterokaryotic mutants

should be caused by the different proportions of the resistant allele in the nuclei, while

the homokaryotic mutants only harbored resistant allele thus showed the high level of

thifluzamide resistance.

Genetic studies of R. solani are difficult to conduct because the fungus rarely

produces sexual or asexual spores, in either the field or the laboratory. Consequently,

the transformation and fusion of protoplasts has become an important tool for the

study of fungal strains with commercial importance.6, 48 The current study used this

approach to generate heterokaryotic isolates by the fusion of hyphae from a

homokaryotic wild-type sensitive isolate and a thifluzamide-resistant mutant. The

resulting progeny confirmed not only that hyphal fusion could produce progeny with

the recombinant genotype but also that the mutated sdhB gene could be used as a

selective marker for further study.

Little is yet known regarding the evolution of resistance to SDHIs in heterokaryotic

fungi such as R. solani, although like all organisms, fungi evolve in response to

selection pressures.42, 49 In current study, it was found that heterokaryotic isolates

could become highly resistant homokaryotic ones both in vitro and in planta, under

the fungicide pressure. These results indicate that heterokaryotic isolates of R. solani


carrying the resistant allele pose a significant risk to the development and evolution of

thifluzamide resistance, which is of great concern given that the thifluzamide resistant

phenotype also exhibits cross-resistance with other SDHIs including mepronil,

fenfuram, carboxin, penflufen, and boscalid.38

A previous study of the SDHI fungicide mepronil found that resistant isolates of R.

solani always exhibit impaired sclerotium production and pathogenicity.4 The current

study found that the CFI of the thifluzamide-resistant mutants showed different fitness

and some resistant strains showed higher CFI than the sensitive wild-types, and

heterokaryons lost their allele under the fungicide pressure with no fitness penalty.

Combined with the evolution risk of heterokaryotic isolates of R. solani, good fitness

increases the resistant risk of thifluzamide in R. solani.

ACKNOWLEDGMENTS

The authors would like to thank Dow AgroSciences for providing the fungicides used

in this study, Prof. Brett Tyler (Oregon State University of USA) for useful advice.

This work was funded by the National High-Technology Research and Development

Programme of China (Grant number: 2012AA101502) and partially supported by the

Special Fund for Agro-scientific Research in the Public Interest of China (Grant

number: 201303023) and the graduate student scientific research innovation projects

of China Agriculture University.


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Table 1. Genotype and thifluzamide sensitivity of R. solani isolates produced by

hyphal fusion and protoplast regeneration.

Strain a Genotype of sdhB at 975

Pyrosequencing EC50 (μg/mL)

C (%) T (%)

JHT158-3 C 100 0 0.057 (0.044-0.069) b T2 T 1.6 98.4 15.631 (11.015-22.084) P97 C 94.2 5.8 0.098 (0.065-0.136) P37 C and T 49.1 50.9 1.601 (1.430-1.846) P45 C and T 32.9 67.1 1.935 (1.689-2.212) P196 C and T 34.6 65.4 2.696 (2.210-3.272) P216 C and T 34.8 65.2 2.731 (2.296-3.246) P229 C and T 35.7 64.3 2.641 (2.186-3.179) P232 C and T 34.0 66.0 2.093 (1.683-2.570) P48 T 2.1 97.9 11.597 (7.866-15.507)

aJHT158-3 and T2 are the parental isolates of the protoplast-regenerated isolates (P97,


P37, P45, P196, P216, P229, P232, P48).

bValues in parentheses are 95% confidence limits.


Table 2 Fitness of sensitive isolates and thifluzamide-resistant mutants of R. solani in the absence of thifluzamide.

Isolate Genotype a Sensitivity b Mycelial growth (cm) c

Mycelial biomass (g) d

Sclerotium weight (g) e

Sclerotium germination (%)

Lesion length (mm) f CFI g

JHT158-3 HM S 8.92a 0.16 abc 0.11 ab 97.92 a 21.47 ab 325.76 c

B310 HM S 7.83 c 0.17 ab 0.11 ab 97.00 a 22.30 ab 305.16 e

FBS-3 HM S 8.46 b 0.16 bc 0.11 ab 98.33 a 21.80 ab 313.52 d

P97 HM S 6.23 f 0.13 cd 0.09 b 92.45 a 20.03 bcd 134.75 j

T2 HM HR 6.80 e 0.12 d 0.11 ab 98.21 a 16.63 cd 143.09 i

P48 HM HR 6.28 f 0.08 e 0.11 ab 97.22 a 15.82 d 83.77 l

P232 HT R 7.65 cd 0.12 cd 0.11ab 100.00 a 21.86 ab 245.19 f

P216 HT R 7.75 cd 0.13 bcd 0.09 b 90.48 b 22.81 ab 195.35 h

P37 HT R 5.82 g 0.07 e 0.10 b 100.00 a 25.76 a 114.08 k

P229 HT R 8.96 a 0.20 a 0.14 a 100.00 a 20.43 bcd 502.18 a

P45 HT R 7.35 d 0.13 cd 0.12 ab 97.22 a 21.14 abc 211.17 g


P196 HT R 7.58 cd 0.14 bcd 0.12 ab 100.00 a 25.37 a 326.02 b

a Genotype: (HM) homokaryotic, (HT) heterokaryotic.

b Thifluzamide sensitivity: (S) sensitive, (R) resistant, (HR) highly resistant.

c Colony diameters measured after 44 h growth on PDA.

d Mycelial dry biomass determined after 54 h growth in PDB.

e Dry weight of sclerotia per PDA plate.

f Lesion lengths on rice sheaths measured 7 days after inoculation under greenhouse conditions.

g CFI (compound fitness index) = mycelial growth × mycelial biomass ×sclerotium formation × sclerotium germination × lesion length.

Means followed by the same letter were not significantly different according to Fisher’s least significant difference test at P = 0.05.

f g


Figure legends:

Fig. 1. Relative expression levels of the sdhB gene in R. solani isolates with different

degrees of thifluzamide resistance in response to different thifluzamide concentrations.

Columns and bars indicate means ± standard deviation. Columns marked with the

same letter are not significantly different by Fisher's protected least significant

difference test (P < 0.05).

Fig. 2. Docking interactions between thifluzamide and the quinone-binding site of the

wild-type SDH and the mutated thifluzamide-resistant SDH. (A) 3D representation of

thifluzamide in the putative binding pocket of the wild-type SDH. (B) 3D

representation of thifluzamide in the putative binding pocket of the mutated SDH.

Key amino acids are represented by cyan sticks, and thifluzamide carbon atoms by

magenta sticks, while hydrogen bonds are represented by blue dotted lines.

Fig. 3. Regression analysis showing the near-linear relationship (R2 = 0.996) between

measured SNP obtained from pyrosequencing and the expected T ratio obtained when

the DNA from plasmids carrying the sensitive (C) and mutant (T) allele were mixed in

different proportions (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10). T

frequencies at position 975 of the R. solani sdhB gene were calculated from the peak

areas as follows: % = peak T/ (peak T + peak C) × 100. Peak areas represent the

averages of two measurements.


Fig. 4. Evolutionary changes in thifluzamide sensitivity of R. solani isolates after

repeated in vitro and in planta subculture in the absence or presence of thifluzamide.

T contents at position 975 of sdhB gene were determined by pyrosequencing. (A & B)

The T contents (%) of R. solani isolates after the first, fifth, 10th, 15th, and 20th

subcultures on PDA containing (A) 0 or (B) 0.05 µg/mL thifluzamide. (C & D) The T

contents (%) R. solani isolates after first, third, fifth, seventh, and 10th subcultures on

rice plants that were sprayed weekly with (C) water or (D) 100.67 µg/mL

thifluzamide.


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